Method for improving nucleic acid synthesis reaction

ABSTRACT

Provided are the following: a method, for improving reactivity of an acid synthesis reaction, comprising a step for adding an ω-amino acid to a reaction solution; a composition, for a nucleic acid synthesis reaction, comprising DNA polymerase, reaction buffer, at least one primer, at least one deoxyribonucleoside triphosphate, and an ω-amino acid; and a reaction buffer, for a nucleic acid synthesis reaction, comprising an ω-amino acid.

TECHNICAL FIELD

The present invention relates to a method for improving reactivity of anucleic acid synthesis reaction, a composition for a nucleic acidsynthesis reaction, and a reaction buffer for a nucleic acid synthesisreaction.

BACKGROUND ART

A nucleic acid synthesis method, in particular a polymerase chainreaction (PCR) method, is a technique for simply amplifying a nucleicacid fragment of interest in a test tube, and recently becomes anessential experimental means not only for genetic research but also in awide variety of fields in biology, medicine, and agriculture.

A nucleic acid synthesis reaction using a DNA polymerase such as PCRoften has a problem with its reactivity such as specificity. In order toavoid a non-specific nucleic acid synthesis reaction, development ofnovel DNA polymerases, improvement of primer design methods,optimization of reaction solution composition, addition of compounds,and the like are attempted. For example, addition of betaine is found tobe effective in amplifying CG-rich templates (Nonpatent Literatures 1and 2).

CITATION LIST Nonpatent Literature

-   Nonpatent Literature 1: “American Journal of Human Genetics”, Sep.    1, 1994, Vol.55, Supplement No.3, p. A238-   Nonpatent Literature 2: “Nucleic Acids Research”, Oct. 1, 1997,    Vol.25, No.19, p. 3957-3958

SUMMARY OF INVENTION Problems to be Solved by the Invention

There have been attempts to improve reactivity of a nucleic acidsynthesis reaction. However, a nucleic acid synthesis reaction may bestill accompanied by a nonspecific reaction or may not synthesize anenough amount of DNA. For these reasons, further improvement inreactivity of a nucleic acid synthesis reaction is needed. An object ofthe present invention is to provide a method for improving reactivity ofa nucleic acid synthesis reaction, a composition for a nucleic acidsynthesis reaction, and a reaction buffer for a nucleic acid synthesisreaction.

Solution to Problems

The present inventors found that reactivity of a nucleic acid synthesisreaction could be improved by addition of an ω-amino acid to a reactionsolution. Thus, the present invention was completed.

That is, the present invention relates to:

-   [1] A method for improving reactivity of a nucleic acid synthesis    reaction, comprising a step of adding an ω-amino acid to a reaction    solution;-   [2] The method according to [1], wherein the ω-amino acid is a    compound represented by the following formula 1:

NH₂—(—CH₂—)_(n)—COOH   [Chemical formula 1]

wherein n is an integer of 2 or more;

-   [3] The method according to [2], wherein n is an integer of 2 to 7;-   [4] The method according to [1], wherein the nucleic acid synthesis    reaction is a polymerase chain reaction;-   [5] The method according to [1], comprising a step of preparing a    reaction solution containing an ω-amino acid, a DNA polymerase, at    least one primer, and at least one deoxyribonucleoside triphosphate;-   [6] The method according to [1], further comprising a step of adding    betaine to the reaction solution;-   [7] A composition for a nucleic acid synthesis reaction, containing:    a DNA polymerase, a reaction buffering agent, at least one primer,    at least one deoxyribonucleoside triphosphate, and an ω-amino acid;-   [8] The composition according to [7], wherein the ω-amino acid is a    compound represented by the formula 1 as defined in [2];-   [9] The composition according to [7], further containing betaine;-   [10] A reaction buffer for a nucleic acid synthesis reaction,    containing an ω-amino acid;-   [11] The reaction buffer according to [10], wherein the ω-amino acid    is a compound represented by the formula 1 as defined in [2];-   [12] The reaction buffer according to [10], further containing    betaine;-   [13] A kit for a nucleic acid synthesis reaction, comprising the    following components:

a DNA polymerase,

a reaction buffering agent,

at least one deoxyribonucleoside triphosphate, and

an ω-amino acid;

-   [14] The kit according to [13], wherein the ω-amino acid is a    compound represented by the formula 1 as defined in [2];-   [15] The kit according to [13], further comprising betaine.

Effects of the Invention

According to the present invention, a method for improving reactivity ofa nucleic acid synthesis reaction, a composition for a nucleic acidsynthesis reaction which has excellent reactivity, and a reaction bufferfor a nucleic acid synthesis reaction are provided.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a result of agarose gel electrophoresis in Example 1.

FIG. 2 shows a result of agarose gel electrophoresis in Example 2.

FIG. 3 shows a result of agarose gel electrophoresis in Example 3.

FIG. 4 shows a result of agarose gel electrophoresis in Example 4.

FIG. 5 shows a result of agarose gel electrophoresis in Example 5.

MODE FOR CARRYING OUT THE INVENTION

The method for improving reactivity of a nucleic acid synthesis reactionof the present invention comprises a step of adding an ω-amino acid to areaction solution. When a nucleic acid synthesis reaction is carried outusing a DNA polymerase in a reaction solution containing an ω-aminoacid, the reactivity of the nucleic acid synthesis reaction can beimproved. The present invention also provides a nucleic acid synthesismethod with excellent reactivity.

The nucleic acid synthesis reaction in the method of the presentinvention is not particularly limited as long as it is a reaction forsynthesizing a DNA complementary to a DNA or RNA template. Examples ofthe nucleic acid synthesis reaction are nucleic acid synthesis reactionswell known in the art, including a primer extension reaction, a reversetranscription reaction, PCR, a reverse transcription polymerase chainreaction (RT-PCR), an ICAN method, a LAMP method, and an SDA method.

When the present invention is used for improvement in reactivity of PCR,general conditions are applicable to thermal cycling conditions for thePCR. For example, PCR is carried out by a reaction consisting of threesteps: melting (denaturation) of a double stranded DNA template intosingle strands, annealing of primers to the single stranded DNAtemplates, and synthesis (extension) of complementary strands from theprimers, or by a two-step reaction, called “shuttle PCR” [see “PCRmethod-Forefront (SaiZenSen)”, Extra issue of “Protein, Nucleic Acid,and Enzyme”, Vol.41, No.5, p. 425-428 (1996)], wherein the annealingstep and the extension step of the above-described three-step reactionare carried out at the same temperature.

As used herein, the improvement in reactivity of a nucleic acidsynthesis reaction means, for example, an effect selected from the groupconsisting of an improvement in reaction specificity and an increase inthe synthesis amount of a DNA having a target nucleotide sequence, butwhich the present invention is not limited to. According to the presentinvention, the reactivity of a nucleic acid synthesis reaction can beimproved by addition of an ω-amino acid to a reaction solution. Whendeduced from the improving effect of ω-amino acids on the reactivity ofa nucleic acid synthesis reaction which has been shown by the presentinvention, ω-amino acids possibly have an ability to improve the primingspecificity of primers, but which the present invention is not limitedto. The improvement in reactivity of a nucleic acid synthesis reactionby an ω-amino acid can be confirmed by, for example, carrying out anucleic acid synthesis reaction with a reaction solution containing anω-amino acid and a nucleic acid synthesis reaction with a reactionsolution containing no ω-amino acid, subjecting the reaction solutionsobtained after the reactions to agarose gel electrophoresis, and thencomparing the reaction specificity or the synthesis amount of a DNAhaving a target nucleotide sequence between the nucleic acid synthesisreactions. The improvement in reactivity of a nucleic acid synthesisreaction by an ω-amino acid can also be confirmed by monitoringamplification of a nucleic acid using an intercalating dye, a FRET(Fluorescence Resonance Energy Transfer) labeled probe or the like, andthen comparing the reaction specificity or the synthesis amount of a DNAhaving a target nucleotide sequence between a nucleic acid synthesisreaction with a reaction solution containing an ω-amino acid and anucleic acid synthesis reaction with a reaction solution containing noω-amino acid. Improvement in the reaction specificity as described aboveis evaluated, for example, based on a reduction in frequency ofnon-specific nucleic acid amplification or a reduction in the synthesisamount of the non-specifically amplified nucleic acid.

As used herein, amino acids are interpreted in a broad sense and alsoinclude amino acids comprising a sulfo group in place of a carboxylgroup. As used herein, the ω-amino acid means an amino acid having aprimary amino group bound to a terminal carbon on the side opposite tothe carbon to which a carboxyl group or a sulfo group is bound, andexcluding a-amino acids. As used herein, the ω-amino acid also includesa β-amino acid, a γ-amino acid, a δ-amino acid, and an ε-amino acid.Examples of the ω-amino acid that can be used in the present inventioninclude, but not limited to, ω-amino acids containing 3 or more carbonatoms, and preferably ω-amino acids containing 3 to 8 carbon atoms.Examples of such an ω-amino acid include compounds represented by thefollowing formula 1:

NH₂—(—CH₂—)_(n)—COOH   [Chemical formula 2]

wherein n is an integer of 2 or more. Among such compounds, preferredare compounds represented by the formula 1 wherein n is an integer of 2to 7.

Examples of the ω-amino acid represented by the formula 1 includeβ-alanine, γ-amino-n-butyric acid (GABA), δ-aminopentanoic acid(5-aminopentanoic acid), ε-aminohexanoic acid (6-aminohexanoic acid),ω-aminoheptanoic acid (7-aminoenanthic acid), and 8-aminooctanoic acid,9-aminononanoic acid, and 10-aminodecanoic acid.

In the method of the present invention, the ω-amino acid is added to areaction solution in an amount effective for improving reactivity of anucleic acid synthesis reaction by a DNA polymerase. The concentrationof the ω-amino acid in a reaction solution which is expected to improvereactivity of a nucleic acid synthesis reaction can be easily determinedby, for example, the above-described method for confirming improvementin reactivity of a nucleic acid synthesis reaction. For example, when6-aminohexanoic acid is used as the ω-amino acid in the method of thepresent invention, the concentration of 6-aminohexanoic acid in areaction solution is preferably less than 300 mM, more preferably 10 to200 mM, and still more preferably 20 to 200 mM, for example 100 mM, butwhich the present invention is not limited to. When β-alanine is used asthe ω-amino acid in the method of the present invention, theconcentration of β-alanine in a reaction solution is preferably 1 M orless, more preferably 100 to 500 mM, and still more preferably 250 to500 mM, for example 500 mM. The suitable concentration range of theω-amino acid in a reaction solution can be easily determined dependingon the kind of a DNA polymerase to be used, the target sequence and thelike, for example, in the same manner as Example 4 or Example 5 asdescribed later.

The reaction solution for a nucleic acid synthesis reaction in themethod of the present invention can be prepared from a combination ofcompositions containing an ω-amino acid, a DNA polymerase, a reactionbuffering agent, at least one primer, at least one deoxyribonucleoside,and a nucleic acid as a template, and the like. As an aspect of thepresent invention, provided is a composition containing a DNApolymerase, a reaction buffering agent, at least one primer, at leastone deoxyribonucleoside, and an ω-amino acid. As another aspect of thepresent invention, provided is a kit containing a DNA polymerase, areaction buffering agent, at least one deoxyribonucleoside triphosphate,and an ω-amino acid. The kit may contain one or more primers.

The DNA polymerase that can be used in the present invention may be anykind of DNA polymerase as long as it has the ability to synthesize a DNAcomplementary to a DNA or RNA template. The DNA polymerase that can beused in the present invention is preferably a heat-stable DNA-directedDNA polymerase. Examples of such a DNA polymerase include heat-stableDNA polymerases derived from bacteria, such as DNA polymerases derivedfrom bacteria of the genus Thermus (DNA polymerases derived from Thermusaquaticus, etc.) and DNA polymerases derived from thermophilic bacteriaof the genus Bacillus (DNA polymerases derived from Bacilus caldotenax,etc.), and DNA polymerases derived from archaea such as DNA polymerasesderived from archaea of the genus Pyrococcus (DNA polymerases derivedfrom Pyrococcus sp., etc.) and DNA polymerases derived from archaea ofthe genus Thermococcus (DNA polymerases derived from ThermococcusKodakaraensis).

As the DNA polymerase, two or more kinds of DNA polymerases may be usedin combination. Examples of two or more kinds of DNA polymerases includecombinations of DNA polymerases having 3′→5′ exonuclease activity andDNA polymerases that do not substantially have 3′→5′ exonucleaseactivity. A technique for PCR with a reaction solution containing suchtwo kinds of DNA polymerases is known as LA-PCR (Long and Accurate PCR).

The amount used of the DNA polymerase in the method of the presentinvention is not particularly limited, and for example, it may be anamount used for a conventional nucleic acid synthesis reaction. For PCR,the amount used of a suitable DNA polymerase is well known to a personskilled in the art. For example, when a DNA synthesis reaction iscarried out in 25 μl of a reaction solution using a DNA polymerase fromThermus aquaticus, the amount of the enzyme contained in the reactionsolution is 0.125 U to 5 U.

The reaction buffering agent as used herein means a compound or amixture having an ability to reduce changes in the hydrogen-ionconcentration (pH) of a reaction solution. A mixture solution of a weakacid and a salt thereof or a weak base and a salt thereof is widely usedas a reaction buffering agent for the purpose of controlling pH, becausethe mixture solution generally has a strong buffering action. In thepresent invention, various reaction buffering agents known in the fieldof biochemistry can be used. Examples of the reaction buffering agentinclude good buffers such as Tris hydrochloric acid, Tris acetic acid,HEPES potassium hydroxide, and HEPES sodium hydroxide, and phosphatebuffers. The pH of the reaction solution in the method of the presentinvention is suitably set in the usual range within which a geneamplification reaction is carried out, for example, in the range of pH8.0-9.5 at 25° C.

The primer is an oligonucleotide having a nucleotide sequencecomplementary to a template nucleic acid. The primer is not particularlylimited as long as it is annealed to the template nucleic acid under thereaction conditions used. When the present invention is used for PCR,two or more primers that can amplify a target sequence and are orientedoppositely to each other may be designed and used. The primer length ispreferably 6 nucleotides or more, more preferably 10 nucleotides or morefrom the viewpoint of specific annealing, and preferably 100 nucleotidesor less, more preferably 30 nucleotides or less from the viewpoint ofoligonucleotide synthesis. The oligonucleotide may be chemicallysynthesized, for example, by a known method. The oligonucleotide mayalso be an oligonucleotide derived from a biological sample. Forexample, the oligonucleotide may be prepared by isolation from adigestion product of a DNA prepared from a natural sample with arestriction endonuclease.

The deoxyribonucleoside is a compound in which deoxyribose is bonded toan organic base and a phosphate group is bonded to the deoxyribose viaphosphoester linkage. Four kinds of deoxyribonucleosides which haveadenine, guanine, cytosine and thymine bases respectively are found innatural DNAs. The adenine, guanine, cytosine and thymine bases are oftenabbreviated to A, G, C and T respectively. The deoxyribonucleosideincludes a free monophosphate type, a diphosphate type and atriphosphate type (i.e., in which the phosphate group is composed ofone, two or three phosphate moieties). It is known that adeoxyribonucleoside triphosphate having hypoxanthine or uracil as thebase can also be used for a nucleic acid synthesis reaction. In thepresent invention, at least one of deoxyribonucleoside triphosphates(e.g., dATP, dCTP, dITP, dGTP and dTTP) and their derivatives is used. Apreferred example of the deoxyribonucleoside triphosphate contained inthe composition of the present invention includes a mixture of fourkinds of deoxyribonucleoside triphosphates, i.e. a mixture of dATP,dCTP, dGTP and dTTP.

In the method of the present invention, the step of adding an ω-aminoacid to a reaction solution may be carried out at any stage duringpreparation of the reaction solution. The step of adding an ω-amino acidto a reaction solution is preferably carried out before incubation ofthe reaction solution at a suitable temperature for a nucleic acidsynthesis reaction. For example, a nucleic acid synthesis reaction maybe carried out after a solution containing a reaction buffering agent (areaction buffer) to which an ω-amino acid is previously added iscombined with a DNA polymerase, at least one primer, at least onedeoxyribonucleoside triphosphate and a template nucleic acid to preparea reaction solution.

Accordingly, the reaction buffer containing an ω-amino acid for anucleic acid synthesis reaction is a preferred aspect of the presentinvention. The reaction buffer may further contain a bivalent cationand/or a monovalent cation, or a salt thereof, and other componentsuseful for a nucleic acid synthesis reaction, in addition to the ω-aminoacid and the reaction buffering agent. Preferred examples of thebivalent cation include bivalent metal ions such as a magnesium ion anda manganese ion. Preferred examples of the monovalent cation include asodium ion, a potassium ion, and an ammonium ion. Preferred examples ofthe other components useful for a nucleic acid synthesis reactioninclude anionic surfactants, nonionic surfactants, andtetramethylammonium salts.

The method of the present invention may further comprise a step ofadding betaine to the reaction solution. The step of adding betaine tothe reaction solution may be carried out at any stage during preparationof the reaction solution. The step of adding betaine to the reactionsolution is preferably carried out before incubation of the reactionsolution at a suitable temperature for a nucleic acid synthesisreaction. The composition of the present invention and the reactionbuffer of the present invention may also contain betaine. The betaine asused herein collectively means compounds having a positive charge and anegative charge at positions that are not adjacent to each other in thesame molecule, in which a dissociable hydrogen atom is not bound to theatom with the positive charge, and having no charge as the wholemolecule. Representative examples of betaine include trimethylglycineand derivatives thereof.

Betaine is known as an additive capable of increasing the reactivity ofa nucleic acid synthesis reaction. The present inventors have found thatan ω-amino acid and betaine produce a synergistic effect on thereactivity of a nucleic acid synthesis reaction. Although a nucleic acidamplification reaction may be inhibited by addition of an ω-amino acidat a high concentration, the present inventors have observed that theinhibition of a nucleic acid amplification reaction is removed byaddition of betaine. The addition amount of betaine in the method of thepresent invention is not particularly limited as long as it is in such arange that an increase in the reactivity of a nucleic acid synthesisreaction by betaine is found, and it is preferably 0.1 M to 3 M, morepreferably 0.3 M to 2.5 M.

The present invention can also be used for real-time PCR in whichamplification of a nucleic acid can be monitored using an intercalatingdye, a FRET labeled probe, or the like. In such a case, the reactionsolution in the method of the present invention, the composition of thepresent invention, and the reaction buffer of the present invention maycontain an intercalating dye, or a FRET labeled probe.

EXAMPLES

Hereinafter, the present invention is more specifically explained byreference to Examples which the present invention is not limited to. Inthe following Examples, TaKaRa PCR Thermal Cycler Dice (registeredtrademark) Gradient (manufactured by TAKARA BIO INC.) was used as areaction device for PCR.

Example 1

After β-alanine (manufactured by SIGMA-ALDRICH) was dissolved inultrapure water (Milli-Q water), Tris acetate buffer (pH 8.9) was addeddropwise to prepare a 5 M β-alanine solution of pH 8.5. In the samemanner, after 6-aminohexanoic acid (manufactured by SIGMA-ALDRICH) wasdissolved in ultrapure water (Milli-Q water), the pH of the solution wasadjusted to prepare a 2 M 6-aminohexanoic acid solution of pH 8.5, andafter 8-aminooctanoic acid (manufactured by SIGMA-ALDRICH) was dissolvedin ultrapure water (Milli-Q water), the pH of the solution was adjustedto prepare a 1 M 8-aminooctanoic acid solution of pH 8.5. Betaine(trimethylglycine; B2629, manufactured by SIGMA-ALDRICH) was dissolvedin ultrapure water (Milli-Q water) to prepare a 5 M betaine solution.These were used in preparation of reaction solutions described below.

PCR reaction solutions for amplification of 987 bp (GC rate: 72.3%) of aTGF β1 gene region were prepared on ice using TaKaRa Ex Taq (registeredtrademark) Hot Start Version (manufactured by TAKARA BIO INC.), 100 ngof Human genomic DNA (manufactured by Clontech Laboratories Inc.) as atemplate, and a primer consisting of a base sequence shown in SEQ IDNO:1 and a primer consisting of a base sequence shown in SEQ ID NO:2 asa primer pair. Four kinds of PCR reaction solutions in total wereprepared which have the reaction solution composition described in theattached instruction of the commercial product, and each of which was 20μL of a reaction solution additionally containing β-alanine at a finalconcentration of 500 mM, 6-aminohexanoic acid at a final concentrationof 100 mM or 8-aminooctanoic acid at a final concentration of 50 mM asthe ω-amino acid, or 20 μL of a reaction solution containing no ω-aminoacid as a control. Furthermore, to the composition of the reactionsolutions thus obtained was added betaine (trimethylglycine) to prepareadditional four kinds of reaction solutions containing betaine at afinal concentration of 300 mM. Next, the PCR reaction solutions thusprepared on ice were subjected to PCR consisting of an activationreaction at 94° C. for 1 minute and then 35 cycles with each cycleconsisting of 98° C. for 10 seconds, 55° C. for 30 seconds and 72° C.for 1 minute. After completion of PCR, 4 μL of each reaction solutionwas subjected to agarose gel electrophoresis to confirm the chain lengthand amplification amount of an amplified product. A result of theagarose gel electrophoresis is shown in FIG. 1. In FIG. 1, M shows alane in which 200 ng of 1 kb DNA Ladder (manufactured by TAKARA BIOINC.) marker was loaded.

As a result, when the PCR reaction solution contained no ω-amino acid, aband corresponding to the desired amplified product of 987 bp was hardlydetected. In contrast, when the PCR reaction solution contained anyω-amino acid, a band corresponding to the desired amplified product of987 bp was detected. When the PCR reaction solution contained betaine inaddition to the ω-amino acid, the amplification amount of the desiredamplified product of 987 bp was increased. When the PCR reactionsolution contained β-alanine or the PCR reaction solution contained6-aminohexanoic acid and betaine, specific amplification of the desiredamplified product of 987 bp was detected.

Example 2

The effect of ω-amino acid was examined in the same manner as Example 1except that Pyrobest (registered trademark) DNA Polymerase (manufacturedby TAKARA BIO INC.) was used instead of TaKaRa Ex Taq (registeredtrademark) (manufactured by TAKARA BIO INC.) and the final concentrationof betaine in the PCR reaction solutions containing betaine was adjustedto 1 M. Pyrobest (registered trademark) DNA Polymerase is a commercialproduct containing a DNA polymerase derived from Pyrococcus sp.

After completion of PCR, 4 μL of each reaction solution was subjected toagarose gel electrophoresis. A result is shown in FIG. 2. In FIG. 2, Mshows a lane in which 200 ng of 1 kb DNA Ladder (manufactured by TAKARABIO INC.) marker was loaded. As a result, when the PCR reaction solutioncontained no ω-amino acid and no betaine, only non-specific amplifiedproducts were detected. In contrast, when the PCR reaction solutioncontained the ω-amino acid, the desired product of 987 bp was detectedas the main amplified product, and in addition, an increase of theamplification amount was also confirmed. When the PCR reaction solutioncontained betaine in addition to the ω-amino acid, non-specificamplification was hardly observed and the desired product of 987 bp wasspecifically amplified. When the PCR reaction solution containedδ-alanine or the PCR reaction solution contained 6-aminohexanoic acidand betaine, the amplified amount of the desired product of 987 bp wasincreased as compared with the PCR reaction solution containing onlybetaine.

Example 3

The effect of ω-amino acid was confirmed in the same manner as Example 1except that a primer pair (a primer consisting of a base sequence shownin SEQ ID NO:3 and a primer consisting of a base sequence shown in SEQID NO:4) for amplifying 965 bp (GC rate: 35.4%) of a Homo sapiens DNA,translocation breakpoint sequences on 22q11: Type C (GenBank:AB261999.1) region was used instead of the primer pair for amplifyingthe TGF β1 gene region.

After completion of PCR, 4 μL of each reaction solution was subjected toagarose gel electrophoresis. A result is shown in FIG. 3. In FIG. 3, Mshows a lane in which 200 ng of 1 kb DNA Ladder (manufactured by TAKARABIO INC.) marker was loaded. As a result, when the PCR reaction solutioncontained no ω-amino acid, non-specific amplification was observed inaddition to amplification of the desired product. In contrast, when thePCR reaction solution contained any ω-amino acid, the non-specificamplification was suppressed.

From the above-described results, it was shown that the addition of anω-amino acid to a PCR reaction solution was effective not only foramplification of a template DNA having a high GC rate, but also foramplification of a template DNA having a low GC rate (a high AT rate)which is difficult to amplify.

Example 4

The concentration of 6-aminohexanoic acid which was effective forimprovement in reactivity of a nucleic acid synthesis reaction wasexamined. Nine kinds of reaction solutions in total were prepared, whichare the same reaction solutions as those of Example 1 except that theycontained 6-aminohexanoic acid at a final concentration of 2 mM, 5 mM,10 mM, 20 mM, 50 mM, 100 mM, 200 mM or 300 mM instead of the variousω-amino acids, and 20 μL of a reaction solution containing no ω-aminoacid as a control. Next, PCR was carried out in the same manner asExample 1, and then 4 μL of each reaction solution was subjected toagarose gel electrophoresis to confirm the chain length andamplification amount of an amplified product. A result of the agarosegel electrophoresis is shown in FIG. 4. In FIG. 4, M shows a lane inwhich 200 ng of 1 kb DNA Ladder (manufactured by TAKARA BIO INC.) markerwas loaded.

As a result, when 6-aminohexanoic acid was added at 20 mM or more, aband probably corresponding to the desired amplified DNA was observed.When 6-aminohexanoic acid was added at 200 mM, only the desired DNA wasamplified.

Example 5

The effective concentration of 6-aminohexanoic acid which was effectivefor improvement in reactivity of a nucleic acid synthesis reaction wasexamined in the same manner as Example 4 except that a primer pair (aprimer consisting of a base sequence shown in SEQ ID NO:3 and a primerconsisting of a base sequence shown in SEQ ID NO:4) for a Homo sapiensDNA, translocation breakpoint sequences on 22q11: Type C (GenBank:AB261999.1) region was used instead of the primer pair for amplifyingthe TGF β1 gene region.

After completion of PCR, 4 μL of each reaction solution was subjected toagarose gel electrophoresis. A result is shown in FIG. 5. In FIG. 5, Mshows a lane in which 200 ng of 1 kb DNA Ladder (manufactured by TAKARABIO INC.) marker was loaded. As a result, when 6-aminohexanoic acid wasadded at 10 mM or more, a band probably corresponding to the desiredamplified DNA was observed. When 6-aminohexanoic acid was added at 50mM, 100 mM or 200 mM, only the desired DNA was amplified.

INDUSTRIAL APPLICABILITY

The present invention is useful in a wide variety of fields includinggene technology, biology, medicine, and agriculture.

SEQUENCE LISTING FREE TEXT

SEQ ID NO:1; Synthetic primer for PCR to amplify of TGF-beta 1 gene.

SEQ ID NO:2; Synthetic primer for PCR to amplify of TGF-beta 1 gene.

SEQ ID NO:3; Synthetic primer for PCR to amplify of translocationbreakpoint sequence region.

SEQ ID NO:4; Synthetic primer for PCR to amplify of translocationbreakpoint sequence region.

1-6. (canceled)
 7. A composition for a nucleic acid synthesis reaction,containing: a DNA polymerase, a reaction buffering agent, at least oneprimer, at least one deoxyribonucleoside triphosphate, and an ω-aminoacid.
 8. The composition according to claim 7, wherein the ω-amino acidis a compound represented by the following formula 1;NH₂—(—CH₂—)_(n)—COOH   [Chemical formula 1] wherein n is an integer of 2or more.
 9. The composition according to claim 7, further containingbetaine.
 10. A reaction buffer for a nucleic acid synthesis reaction,containing an ω-amino acid.
 11. The reaction buffer according to claim10, wherein the ω-amino acid is a compound represented by the followingformula 1;NH₂—(—CH₂—)_(n)—COOH   [Chemical formula 1] wherein n is an integer of 2or more.
 12. The reaction buffer according to claim 10, furthercontaining betaine.
 13. A kit for a nucleic acid synthesis reaction,comprising the following components: a DNA polymerase, a reactionbuffering agent, at least one deoxyribonucleoside triphosphate, and anω-amino acid.
 14. The kit according to claim 13, wherein the ω-aminoacid is a compound represented by the following formula 1;NH₂—(—CH₂—)_(n)—COOH   [Chemical formula 1] wherein n is an integer of 2or more.
 15. The kit according to claim 13, further comprising betaine.